The broad, long-term objectives of this project are to (A) describe at a molecular level how cyclin dependent protein kinases (Cdks) are regulated to control critical cell cycle transitions, (B) understand how loss of these controlling mechanisms leads to genetic aberrancies, and (C) develop means of correcting or suppressing defects resulting from deficiencies in cell cycle control mechanisms. This proposal will specifically focus on aspects of the function and turnover of Sic1, an inhibitor of specific cyclin-dependent kinases of S. cerevisiae and the interaction of Sic1 with Cdc28. The specific aims are to (l) test the hypothesis that S phase is initiated by proteolytic turnover of Sic1 protein and that this turnover is dependent upon the activity of Cdc28 protein kinase and Cdc34 ubiquitin conjugating enzyme; (2) test the hypothesis that Sic1 is ubiquitinated in a Cdc28 and Cdc34 dependent manner, (3) delineate potential subdomains of the Sic1 protein with respect to its in vivo stability and its ability to bind, inhibit and be phosphorylated by Cdc28 protein kinase, and (4) identify sites on Cdc28 responsible for Sic1 binding. Sic1 is an inhibitor of specific cyclin dependent kinases (Cdks) in yeast and a suppressor of ras transformation in animal cells. Other proteins with Cdk inhibitory activity include a potent tumor suppressor (MTS1/INK4) and a key target of the p53 tumor suppressor (CIP1/WAF1). Further understanding of how this class of proteins functions promises to open up new genetic and chemotherapeutic approaches to cancer treatment. This application proposes to delineate the role of Sic1 in the coordination of events in the G1 period of the yeast cell cycle and to identify the sites of Sic1 interaction with the Cdk. The research design will take advantage of the genetic techniques available in yeast and the wealth of mutations in cell cycle related processes to achieve the specific aims. Degradation rates of the Sic1 protein will be ascertained using strains defective in specific cell cycle events, with a focus on alleles of CDC28 and CDC34. Phosphorylation and ubiquitination of Sic1 protein will also be assayed in vitro with both wild type and mutant cell extracts. Mutational screens will be carried out that will allow the identification of Sic1 mutations that are defective in turnover and Cdk inhibition and of CDK mutations that are defective in Sic1 recognition.